Method of separating live viruses from nonviral protein matter



Y ers be-conic sen Patented May 2, 1 950 g tics scram-arms Live vthuehs FEQMMQNY EAIEn TEEN egaldfi. gag, and Stewart Ai st on, Pearl ignors to American Gyaliainicl (-lompany,"-New orlg, N. Y., a corporation lof 'Maine .Dremi g- A lication @Qcwhg ,lggii,

. 'This i-nvention relates to improvements in the art of preparing vaccines and "antigens and in particular to a new process by which viruses ngay be separated frorn associated pnotein'a natter to such 'egitent that the purified {viruscan be used in a yaecinesuitable for injection into huzn-anzbe' Numerous investigators have shown that the administration of active or" i nacti-ve virus produces an increase in th titer of the circulating antibodies specific against the disease and that the average-antibodyrespcnse of human beings is *directly'related, though not directlyproportionail, mane amountof virus administered. Eihe commercial production of such vaccines depends upon the ability to successtully produce large amounts .of the .virus in .a form sufiiciently free fron foreig-n ,prqteins .to injected into human beings without inducing untoward reactions.

Viruses are unique-in that they must be props t-rerun. 700,372 \fifilaims. ,(ol. mag-as agated in the presence of living tissue. Onefof f the most common methods of propagation is in incubated'afertile eggs from which the .virus is s1ti zed "po fi injection-f et vac,- cines containing egg protein, it is necessary that methods be devised by --rvhich-,the sensitizing protein can be separated from the virus without serig'gus loss. ,of virus ;or decrease in its antigenic prqpeigties. vfirus es have also been propagated in .ahimals @1 9 the meta -spleen l ve and 9. 1 2

organs, rthe gblgod itself, have been removed and'vaccines prepared therefrom. -;I sue ca es it is also desirable 'to remove non V p n matter tonaizoidthe sensitizationof pers n re ceiving the vaccine.

.Narious-methods .havecbeendevisedato-rconcentrate virusesfland .to eliminate foreign .proteins as fl-e iflm sa e an m re h i ht intenrat on .te he use neone h me h in phzes :x neat d vima ne the; :zbulk 'pof'rrthe active, ,yi-rus then precipitates. 1

The virus has also been adsorbed ,on various adsonbentspincluding: ,red blood cells; ,calciurn -volved--the. use of thigh. speed centrifuges, :it she-1 ing claimed- :that infectious. allantoi'c'fluid which:

pfiosphateyaluminum; zinc and magnesium hy shows a protein contentof less than 10% of-virus protein and over 90% .ot low n olecular weight host proteins can be pur-i-fied to a product which the non-virus protein matter is less than about 5% of the total.

Although s m ,o these methods hav been in commercial ye the purification of virus vaccines they are suhicetto numerous disadvan tages, the most serious being the difficulty in- ;volved in preparing products substantially" free of non-viral proteins. Another serious (1' ta ge is the fact that thereeeverytf nns tively low}! The centrifuge method, although probably the es available, hasithe' additional disadvantages inthat because of the *high speeds and'low output ofthe machines, they are quite subject" to meon-anneal breakdowns and yield a relatively low'volumeof-rthevaccine:

I'n attenipting tofifibetfif methods of sepa rating influenza virus from the non-viralgirotein matter associated therewith when the virus is propagated-i gg's, -we -hav'e discovered that the virus canbe precipitated from aqueous sus- {to prepanea vaccine withoutJa-nysubstantiahiQssof'virus and whichis'fre'er trqiy ext a equs p teins of ,ncn-virgal type than any .of theo ea" et hpw ret the sehti i iheorderthat the nature of the-invention-will gee-more apparentcertain' procedures will be descubed- 51 3? the e L e s @1 25 teta h 1 I h seazi sesur e-tse se; e s rh ihe'cihoeh at e thr ugh a em theshell a orethe ai a. 0f elevenday old Wm The infected embryo F.-:-;:Eor Alto-$8 trations of. and' 0" zper'cent were ob,-

tainedforthelvariousspontions respectively. :Tlietemperature of the al-lantoic fluidaalcohol mix,- 'ture was gradually towered -=the -,c0ncent1fazv ('.:tion ,oiqalcuholx iincteased so that the temliterature of the mixture was held at 5 C. during the later stages of alcohol addition. The mixture was allowed to stand at 5 C. for approximately 3 hours and then was centrifuged at 3500 R. P. M. for 30 minutes in an international size 1 centrifuge equipped with an angle head rotor. This centrifugation was carried out at 5 C. The supernatant was discarded. The precipitate was resuspended in ml. of 0.1 molar phosphate bufier of pH 7.0 and allowed to stand at room temperature for 1 hour. The suspension was centrifuged a second time at 2,000 R. P. M. for minutes in the same type of centrifuge except that this time the centrifugation was done at room temperature.

The supernatant fluid, which could be easily decanted from the insoluble protein sediment, was tested to determine the amount of virus present in terms of CCA activity. The results shown in Table 1 indicate that a yield of 92 per cent of virus was achieved by using a final alcohol concentration of 25 percent, while alcohol concentrations of and 30 percent gave yields of 51 and 58 percent, respectively.

Table 1 Percent concentration of g g; ethyl alcohol a Weiss allantoic fluidstarting materi 67 Weiss allantoic fluid, 2x concen- By similar procedures it was determined that an 85 to 95 percent recovery of Lee virus was obtained by a final concentration of percent ethanol.

Table 2 shows the results obtained when methanol was added to Lee allantoic fluid under conditions similar to those described above.

It is seen that a 100 percent yield of virus was obtained by using 25 percent methanol and that recoveries'of 98 and 96 percent were obtained by using methanol concentrations of 30 and'20 percent, respectively. Furthermore, it is apparent that the optimal concentration of methanol is not as critical as that of ethanol and that excellent yields of virus may be obtained over a fairly above, while greatly reduced in non-viral protein content, still contained more chick protein than desired. While this residual chick protein may be removed by repeated precipitations with alcohol, we have found it advantageous to remove further amounts at this stage of the process by centrifugation. The following procedure is illustrative.

To 1500 ml. of infected allantoic fluid, containing no preservative, were added 500 ml. of absolute methyl alcohol as described above. The mixture was held at 5 C. for 3 hours and then centrifuged at 3500 R. P. M. for 30 minutes, the centrifugation procedure taking place in a 5 C. chillroom. The supernatant was discarded. The precipitate was resuspended in 200 ml. of 0.1 molar phosphate bufier of pH 7.0. After holding for 1 hour at room temperature, the insoluble.

protein material was removed by centrifuging at 2,000 R. P. M. for 15 minutes at room temperature. The supernatant thus was concentrated 7 times in terms of the original starting material. The supernatant was then batch-bowled in a, Sharples laboratory centrifuge for 30 minutes at 50,000 R. P. M. and then 1 liter of 0.1 molar phosphate buffer of pH 7.0 was run through the bowl at the rate of 1 liter per hour, maintaining the speed at 50,000 R. P. M. The contents of the bowl were then shaken to resuspend the virus in the phosphate buffer and the final volume Wasbrought to 300 ml. thus effecting a five fold concentration in terms of the original allantoic fluid. 1 Table 3 shows the results obtained with asample of Lee strain, allantoic fluid processed as described above.

Table 3 Total N GOA Per g g in mgs. units er Cent per ml mg. Yield Lee allantoic fluid 102 0. 127 Lee allantoic fluid, Alc. ppt.,

cone. 7 l/2r 735 0.236 8, 97 Lee allantoic fluid, Ale. ppt., centrifuged 5r conc 485 0.033 14, 700

In the following table are shown the results obtained by the identical procedure with the Weiss strain.

The relative infectivity of the methanol precipitated virus was determined and found to be unimpaired even after three successive precipitations.

trated 10- Obviously, various modifications may be made 1n the procedure just described. Acetone and. 3 other known protein precipitants may be used in Repeated precipitations can also be conducted under different conditions of i place of alcohol.

temperature, alcohol concentration, pH and ionic strength whereby a vaccine substantially free of non-viral protein matter-can beobtained. These The final product possessed an infectivity endpoint of l0 While the control material tivirus.

freezin Point -.of the solutwn .to abbo ionicsstrength from 0.005 to 0,5 ands] ch31 .09 1.-

centrationirom .15-% to about ,a0;% ,by-.volum e.

Astthesevar-iant factors are dependentnponeach other to some extent, soptimum conditions for maximum puriiication are determined by simple experimentation --within rthe range. Under ordinary conditions the aqueous suspension of virus as prepared fmm the {living tissue ;will not need any particular adjustmentof pH or ionic strength inasmuch as it will have these values withinthe desired range. When the crude virus preparatron -eontains much tissue it may sbe --removed at any stage of theeprocess:bylow-speedcentritugation -o'r {by other suitable methods. :One such process 'i I i'v-olves suspending the virus :material after initial alcohol precipitation at /se its origina-l'vohim'e at about pH- SQ iwimooz :molarphosphate buffer solution. 'Oncentrifugingat :re'lia tively l'ow :speeds: theatissue and :much :ofthe iereign eprotein stays in suspension while :the virus is sedimented. This process makesdt r1303 sible to efiect aseparatiombetween viral-.andnonviral proteins. The sedimented :virus fol-lowing this cprocerlure m'ayibe then .res-uspended in rappropria'tezbufter solution such as. O;l molanphosphate buffer of pH 7.0.

Although, as noted above, separation of nonviral proteins can be achieved by repeated precipitation with alcohol the most efiective method appears to involve the combined use of alcoholic precipitation and centrifugation. By this combination of steps it is possible to separate protein fractions that cannot be conveniently separated by either alcoholic precipitation or centrifugation procedures alone. It appears that some of the non-viral protein matter possesses some of the same sedimentation characteristics as the viral protein and on centrifugation complete separation is difiicult or impossible. The alcohol precipitation characteristics of the dilTerent kinds of protein matter difier sufficiently, however, to make it possible to separate them by this procedure-that is, by alcohol precipitation and fractionation methods.

Obviously the process described hereinabove is applicable to the preparation of both immunizing vaccines and diagnostic antigens. The exact details of the preparation of these products are matters well within the skill of the art. The particular vehicle in which it is to be dispersed and the titer of the final product are open to choice. Inactivation of the virus by formaldehyde or other means, if desired, is also a matter outside the scope of the present invention.

The procedure described herein for influenza virus may be applied to the production of purifled vaccines and antigens of other viruses and Rickettsiae including those such as eastern and western equine encephalomyelitis, yellow fever, St. Louis encephalitis, Colorado tick fever, rabies, psittacosis, Rocky Mountain spotted fever, epidemic and murine typhus, scrub typhus, Q fever, Jap B encephalitis, poliomyelitis and Newcastle Inasmuch as viruses and Rickettsiae possess many of the same fundamental characteristics and are often classified together, the term virus as used herein and in the claims is employed in a generic sense to include the above and other related viruses and Rickettsiae falling within the scope of the present invention.

We claim:

1. A method of separating live viruses from non-viral protein matter in association therewith 9 423 brand i ee iron c nrecipitated-adser and thereafter separa ng the prep fest ve iru mth 1 ai sql tion rmcs li ebn s a matter. -2 A method .Q se aratin .i tvi ra r tei tm tt t outde tnu ion. s to iniecuv v s aiba iru which;c inn sesrth step to sslowlv d to a-q d ue ussuspens ont nte ni s 1 and -.non -r ;pro ein'matie .andh vi withi athfi ran 5. 1m Mae a i n; of. s vreenr flfl tan 05 a h J v. a l ifi u li elk? alcohol unaided by and free from co-precipitated adsorbing agents and thereafter separating the precipitated infective virus from the aqueous phase which retains in solution most of the non-viral protein matter.

3. A method of separating live viruses from non-viral protein matter in association therewith without destruction or loss of infectivity of the virus which comprises the steps of slowly adding to a cold aqueous suspension containing live virus and non-viral protein matter and having a pH within the range 5.0 to 8.0 and an ionic strength of between 0.005 and 0.5 at such rate as to avoid denaturation of said virus from about 15% to 40% by volume of ethanol while maintaining the temperature of the suspension below 5 C. whereby direct precipitation of the virus is caused to take place by the action of said alcohol unaided by and free from co-precipitated adsorbing agents and thereafter separating the precipitated infective virus from the aqueous phase free from inorganic salts and most of the non-viral protein matter originally associated therewith.

4. A method of separating live viruses from non-viral protein matter associated therewith in chorioallantoic fluid without destruction or loss of infectivity of the virus which comprises the steps of slowly adding to a cold aqueous suspension of chorioallantoic fluid containing live virus and non-viral protein matter and having a pH within the range 5.0 to 8.0 and an ionic strength of between 0.005 and 0.5 at such rate as to avoid denaturation of said virus from about 15% to 40% by volume of methanol while maintaining the temperature of the suspension below 5 0. whereby direct precipitation of the virus is caused to take place by the action of said alcohol unaided by and free from co-precipitated adsorbing agents and thereafter separating the precipitated infective virus from the aqueous phase which retains in solution most of the non-viral protein matter.

5. A method of preparing vaccines which comprises the steps of slowly adding to a cold aqueous suspension containing infective virus and non-viral protein matter and having a pH with in the range 5.0 to 8.0 and an ionic strength between 0.005 and 0.5, 15% to 40% by volume of methanol at such rate as to avoid denaturation of said virus, while maintaining the temperature of the suspension below 5 C. whereby the virus is precipitated by the action of said methanol unaided by and free from co-precipitated adsorbing agents and without loss of infectivity, separating the precipitated virus from the aqueous phase, resuspending the precipitated virus in an aqueous solution, centrifuging the solution and recovering the aqueous supernatent liquid containing the infective virus substantially free from non-viral protein matter.

-6. A method of preparing an influenza vaccine comprising th steps of slowly adding 15% to 40% by volume of methanol to a cold aqueous suspension of chorioallantoic fluid containing ."live influenza virus and having a pH between 5.0 :and 8.0 and an ionic strength from about 0.005 to 0.5 at such rate as to avoid denaturation of said influenza virus and while maintaining the temperature of the suspension below 5 0. whereby the influenza virus is precipitated with undiminished virulence, centrifuging the alcoholic suspension to sediment the precipitated virus, separating the precipitated virus in the supernatant aqueous phase, resuspending the precipitated virus in an aqueous suspension at about pH 7.0.

centrifuging the u pension and recovering the supernatant aqueous phase containing the live influenza virus, supercentrifuging the aqueous: phase and recovering the sedimented live innitenza virus substantially free of non-viral protein" matter and thereafter resuspending the sedimerited virus in aqueous solution. HERALD R. COX. STEWART AISTON.

REFERENCES CITED Letter of Aug. s, 1947, from U. s. P. H. s. con- 1 cerning the article by Topping (1 page).

Practical Physiological Chemistry, by Hawk' et al., 12th ed. Copyright 1947, The Blakiston Co.,- pages 157 and 158. U. S. Public Health Reports, article by Top-:; ping et al., pages 7'78 to 781, May 31, 1946.

Comptes Rendus (Doklady) de IAcademie des; Sciences de IURSS, article by Tovarnizky et al.,- pages 194-195.

Science, article by Taylor et al., pages 587 to 589, Dec. 31, 1943. r

Journal of the Franklin Institute," article by McKinstry, pages 310 to 316, volume 238, Oct; 1944. 

1. AN METHOD OF SEPARATING LIVE VIRTUSES FROM NON-VIRAL PROTEIN MNATTER IN ASSOCIATION THEREWITH WITHOUT DESTRUCTION OR LOSS OF INFECTIVITY OF THE VIRUS WHICH COMPRISES THE STEPS OF SLOWLY ADDING TO A COLD AQUEOUS SUSPENSION CONTAINING LIVE VIRUS AND NON-VIRAL PROTEIN MATTER AND HAVING A PH WITHIN THE RANGE 5.0 TO 8.0 AND AN IONIC STRENGTH OF BETWEEN 0.005 AND 0.5 AT SUCH RATE AS TO AVOID DENATURATION OF SAID VIRUS FROM ABOUT 15% TO 40% BY VOLUME OF A LOWER ALIPHATIC ALCOHOL WHILE MAINTAINING THE TEMPERATURE OF THE SUSPENSION BELOW 5*C. WHEREBY DIRECT PRECIPITATION OF THE VIRUS IS CAUSED TO TAKE PLACE BY THE ACTION OF SAID ALCOHOL UNAIDED BY AND FREE FROM COPRECIPITATED ADSORBING AGENTS AND THEREAFTER SEPARATING THE PRECIPITATED INFECTIVE VIRUS FROM THE AQUEOUS PHASE WHICH RETAINS IN SOLUTION MOST OF THE NON-VIRAL PROTEIN MATTER. 